Ribonucleases catalyze the hydrolysis of the P-O5? bond in RNA. This reaction occurs in two steps: transphosphorylation of RNA to a 2?,3?-cyclic phosphodiester intermediate and hydrolysis of this intermediate to a 3?-phosphomonoester. 31P NMR spectroscopy was used to monitor the accumulation of the 2?,3?-cyclic phosphodiester intermediate during the transphosphorylation and hydrolysis reactions catalyzed by various ribonucleases and by small molecules. The intermediate was found to accumulate during catalysis by monomeric bovine pancreatic ribonuclease A (RNase A), a dimer and a trimer of RNase A, bovine seminal ribonuclease, RNase T1, barnase, and RNase I. These enzymes, which are of widely disparate phylogenetic origin, released rather than hydrolyzed most of the intermediate formed by transphosphorylation of RNA. In contrast, the intermediate did not accumulate during catalysis by hydroxide ion or imidazole buffer. In the presence of these small molecules, hydrolysis is faster than transphosphorylation. A trapping experiment was used to assess the throughput of the reaction catalyzed by RNase A. [5,6-3H]Uridylyl-3?-5?)adenosine was incubated with RNase A in the presence of excess unlabeled uridine 2?,3?-cyclic phosphodiester, which dilutes the specific radioactivity of any released cyclic intermediate. Only 0.1% of the RNA substrate was found to be both transphos-phorylated and hydrolyzed without dissociating from the enzyme. These results suggest that ribonucleases have evolved primarily to catalyze RNA transphosphorylation and not RNA hydrolysis.